Expression of chimeric polypeptide with variable lymphocyte receptors on immune cells and uses for treating cancer

ABSTRACT

This disclosure relates to recombinant cellular expression of chimeric proteins with peptide sequences derived from lymphocyte receptors and uses for treating cancer. In certain embodiments, the disclosure relates to a recombinant vector comprising a nucleic acid that encodes a chimeric protein with a segment with a targeting moiety based on a variable lymphocyte receptor (VLR) capable of binding a tumor associated antigen and a segment with a T cell signal transduction subunit. In certain embodiments, the recombinant vectors are used in immune based cancer treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/117,811 filed Aug. 10, 2016, which is the National Stage of International Application No. PCT/US2015/014975 filed Feb. 9, 2015, which claims the benefit of priority to U.S. Provisional Application No. 61/938,057 filed Feb. 10, 2014. The entirety of each of these applications is hereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED VIA THE OFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 14078USDIV_ST25.txt. The text file is 35 KB, was created on May 1, 2019, and is being submitted electronically via EFS-Web.

BACKGROUND

Chemotherapy is the standard of care for the treatment of many types of cancer, and alternatives methods for treating cancer are need in situations where chemotherapy is not effective.

The human immune system is sometimes able to prevent or slow the growth of cancerous cells through recognition by T cells. In order to improve the ability of immune cells to kill cancerous cells, T cells can be isolated from the blood of a patient and genetically altered to specifically bind proteins expressed on the surface of cancerous cells. When put back into the patient, the modified cells more efficiently target the cancerous cells. CD19 is a protein expressed on cancerous B cells. Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177):177ra38.

Chemotherapy agents typically act by killing cancerous cells but they also affect other circulating cells such as T cells. Dasgupta et al. report engineering immune cells to be resistant to cancer drugs in order to prevent T cell death and enhance tumor cell killing during chemotherapy. Biochem Biophys Res Commun, 2010, 391(1):170-5.

Humans generate T- and B-cell antigen receptors primarily by the assembly of Ig V-(D)-J gene segments and somatic hypermutation. Lampreys and hagfish have an alternative system that is based on variable lymphocyte receptors (VLRs), the diversity of which is generated from leucine-rich repeat (LRR) cassettes. Yu et al., report purification and identification of cell surface antigens using lamprey monoclonal antibodies. Immunol Methods, 2012, 386(0): 43-49. See also Yu et al., A lamprey monoclonal VLR antibody recognizes a novel plasma cell antigen, The J of Immunol, 2013, 190, Abstract 114.11; Han et al. Antigen recognition by variable lymphocyte receptors, Science, 2008 321:1834-183; Hirano et al., The evolution of adaptive immunity in vertebrates, Adv Immunol, 2011, 109:125-57; WO 2013/078425; US 2011/0230374; WO 2010/065407; and WO 2008/016854.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to recombinant cellular expression of chimeric proteins with peptide sequences derived from lymphocyte receptors and uses for treating cancer. In certain embodiments, the disclosure relates to a recombinant vector comprising a nucleic acid that encodes a chimeric protein with a segment with a targeting moiety based on a variable lymphocyte receptor (VLR) capable of binding a tumor associated antigen and a segment with a T cell signal transduction subunit. In certain embodiments, the recombinant vectors are used in immune based cancer treatments.

In certain embodiments, the recombinant vectors comprise a nucleic acid that encodes a chimeric polypeptide comprising a targeting sequence of variable lymphocyte receptor domain or variant thereof, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain such as CD3zeta (CD3Z).

In certain embodiments, the variable lymphocyte receptor domain contains a polypeptide sequence of less than 250 amino acids and 4 or 5 or more segments having the sequence XXLXLXX (SEQ ID NO: 1) wherein X may be any amino acid and L may be, individually and independently at each occurrence, leucine or isoleucine or optionally one L (leucine or isoleucine) may be substituted with any amino acid.

In certain embodiments, the variable lymphocyte receptor has a sequence VXCXXXXL XSVPAXIPTTTXXLXXXXNXITKXXPGVFDXLXXLXXXXLXXNXLXXXPXGXFD (SEQ ID NO: 2) wherein X may be any amino acid.

In certain embodiments, the variable lymphocyte receptor has an amino acid sequence disclosed herein such as SEQ ID NO: 4, 6, 8, 10, or variant or a sequence with greater than 80, 85, 90, 95% identity thereto.

In certain embodiments, the nucleic acid sequence has a nucleotide sequence disclosed herein such as SEQ ID NO: 3, 5, 7, or 9 or variant or a sequence with greater than 80, 85, 90, 95% identity thereto.

In certain embodiments, the costimulatory molecule is selected from CD28, CD80, CD86 or fragment or variant.

In certain embodiments, the signal-transduction component of the T-cell antigen receptor comprises an immunoreceptor tyrosine-based activation motif with the consensus sequence YXXLXXXXXXXXYXXL (SEQ ID NO: 15) wherein X is any amino acid L is leucine or isoleucine and optionally one or two X are optionally deleted.

In certain embodiments, the recombinant vector further comprises an interleukin sequence such as IL-2 or fragment or variant.

In certain embodiments, the recombinant vector further comprises CD8 or fragment or variant.

In certain embodiments, the recombinant vector further comprises a nucleic acid encoding an enzyme that confers resistance to cellular damage in the presence of a chemotherapy agent.

In certain embodiments, the recombinant vector further comprises a nucleic acid encoding methylguanine methyltransferase (MGMT), dihydrofolate reductase (DHFR), cytidine deaminase (CD), and multidrug resistant protein (MDR-1) or variants thereof.

In certain embodiments, the recombinant vector further comprises a nucleic acid encoding the variable lymphocyte receptor sequence that specifically binds to a tumor associated antigen such as CD5, CD19, CD20, CD30, CD33, CD47, CD52, CD152(CTLA-4), CD274(PD-L1), CD340(ErbB-2), GD2, TPBG, CA-125, CEA, MAGEA1, MAGEA3, MART1, GP100, MUC1, WT1, TAG-72, HPVE6, HPVE7, BING-4, SAP-1, immature laminin receptor, vascular endothelial growth factor (VEGF-A) or epidermal growth factor receptor (ErbB-1).

In certain embodiments, the disclosure relates to isolated cells comprising the recombinant vectors disclosed herein.

In certain embodiments, the isolated cells are selected from T helper cell, cytotoxic T cell, natural killer T cell, or γδ T cell.

In certain embodiments, the disclosure relates to methods of treating cancer comprising isolating immune cells, e.g. T cells, gamma delta T cells, or NK cells, and mixing with or transferring a recombinant vector disclosed herein into the cells under conditions such that the recombinant vector expresses in the isolated cells a chimeric polypeptide comprising a variable lymphocyte receptor domain or variant, a transmembrane molecule domain, a T cell costimulatory molecule domain, and a signal-transduction component of the T-cell antigen receptor domain providing modified immune cells, T cells, gamma delta T cells, or NK cells; and implanting the modified immune cells, T cells, gamma delta T cells, or NK cells into a subject in need thereof.

In certain embodiments, the recombinant vector encodes an enzyme that confers resistance to cellular damage in the presence of a chemotherapy agent, and an effective amount of the chemotherapy agent is administered to the subject before, during, or after implanting the cells into the subject.

In certain embodiments, the isolated immune cells, T cells, gamma delta T cells, or NK cells are isolated from the subject to receive the implanted modified cells.

In certain embodiments, the cancer is selected from neuroblastoma, glioblastoma, glioma, breast cancer, prostate cancer, colon cancer, lung cancer, skin cancer, renal cancer, ovarian cancer, pancreatic cancer, stomach cancer, leukemia, lymphoma, or melanoma.

In certain embodiments, the disclosure contemplates recombinant polypeptides, recombinant vectors comprising nucleic acids encoding polypeptides reported herein and expression systems for producing those polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a nucleic acid (SEQ ID NO: 3) that encodes variable lymphocyte receptor that binds to neuroblastoma cells (VLR clone 4).

FIG. 1B shows the amino acid sequence (SEQ ID NO: 4) of the variable lymphocyte receptor translated by the nucleic acid in FIG. 1A.

FIG. 1C illustrates a nucleic acid (SEQ ID NO: 5) that encodes variable lymphocyte receptor that binds to neuroblastoma cells (VLR clone 18).

FIG. 1D shows the amino acid sequence (SEQ ID NO: 6) of the variable lymphocyte receptor translated by the nucleic acid in FIG. 1C.

FIG. 1E illustrates a nucleic acid (SEQ ID NO: 7) that encodes variable lymphocyte receptor that binds to neuroblastoma cells (VLR clone 19).

FIG. 1F shows the amino acid sequence (SEQ ID NO: 8) of the variable lymphocyte receptor translated by the nucleic acid in FIG. 1E.

FIG. 2A shows is a codon optimized VLR sequence (SEQ ID NO: 9) that encodes a protein shown to bind to CD5, the CAR expressing this protein can be used to treat T cell malignancies.

FIG. 2B shows amino acid sequence (SEQ ID NO: 10) of the variable lymphocyte receptor translated by the nucleic acid in FIG. 2A.

FIG. 3 illustrates a method of generating VLR-CAR sequences. Tumor samples or tumor cell lines (e.g., neuroblastoma, B cells, T cells), are isolated from a patient e.g., diagnosed with leukemia, and injected into a lamprey. Tumor specific VLRs are identified and cloned into a chimeric antigen receptor cassette within a lentiviral vector. SP refers to the signal peptide and LTR refers to the long terminal repeat. The vector is transduced into immune cells (T cells, natural killer cells) and re-introduce into the same or a different patient.

FIG. 4A shows data from the screening process using yeast display assays for the isolation of an anti-tumor VLR specifically for neuroblastoma. MACS sorted neuroblastoma are provided in the top right quadrant (FACS sort). Colonies are plated on agar, sequenced and cloned into CAR cassette.

FIG. 4B shows data evidencing T cell activation after transduction using the lentiviral vector containing the cloned cassette of FIG. 4A.

FIG. 5 shows a schematic of the lentiviral vector used to transduce T cells and to measure VLR-CAR expression. Antibodies to the Myc tag are used to show cell surface expression of the VLR-CAR.

FIG. 6A shows a cell count vs Myc tag expression (i.e. cell surface VLR-CAR expression) of naïve Jurkat cells by FACS.

FIG. 6B shows data wherein Jurkat cells were transduced at an MOI 2 with lentiviral vector shown in FIG. 5 and wherein genetically modified cells were identified for chimeric antigen receptor expression using the Myc tag.

FIG. 6C shows data wherein Jurkat cells were transduced at an MOI 10 with lentiviral vector shown in FIG. 5 and wherein genetically modified cells were identified for chimeric antigen receptor expression using the Myc tag.

FIG. 7A shows naïve Jurkat cells co-cultured with a BCL cells, and T cell activation is monitored by CD69 expression.

FIG. 7B shows BCL-VLR-CAR transduced Jurkat cells co-cultured with a BCL cells, transduced at MOI 2. Transduced Jurkat cells were incubated with the BCL cell line expressing the target B-cell receptor and monitored by CD69 expression as a measure of T cell activation.

FIG. 7C shows BCL-VLR-CAR transduced Jurkat cells co-cultured with a BCL cells, transduced at MOI 10, and monitored by CD69 expression as a measure of T cell activation.

FIG. 8A shows CD5-VLR-CAR transduced Jurkat cells activated by CD5 expressing cells (as measured by CD69 expression).

FIG. 8B shows cells expressing GFP (control) instead of the CD5-VLR-CAR, similarly transduce as in FIG. 8A, are not activated as monitored by CD69 expression.

FIG. 9A illustrates an embodiment of this disclosure.

FIG. 9B illustrates an embodiment of this disclosure.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

The terms “protein,” “peptide,” and “polypeptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. A heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species. For example, a chimeric polypeptide comprising a targeting sequence refers to a fusion protein in which the targeting sequence is linked to a different polypeptide not associated with the naturally occurring protein from which targeting sequence is derived. A chimeric polypeptide refers to covalent linkage of two distinct polypeptides heterologous to each other. The linkage can be by chemical or recombinant means, for instance. In some cases, the linkage is chemical, wherein a reaction between the antibody moiety and the fusion partner has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included, e.g., between the targeting sequence and the heterologous polypeptide. The terms “variant” and “mutant” when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. Variants may be in the form of functioning fragments that may be greater than 25, 50, or 100 amino acids and in some instances less than 100, 150, or 200 amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have “non-conservative” changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).

The term “nucleic acid” refers to a polymer of nucleotides, or a polynucleotide, as described above. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements, as described below.

The term “a nucleic acid sequence encoding” a specified polypeptide refers to a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide, polynucleotide, or nucleic acid may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present disclosure may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term “recombinant” when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant nucleic acid molecule. The term recombinant nucleic acid is distinguished from the natural recombinants that result from crossing-over between homologous chromosomes. Recombinant nucleic acids as used herein are an unnatural union of nucleic acids from nonhomologous sources, usually from different organisms.

The terms “vector” or “expression vector” refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

Protein “expression systems” refer to in vivo and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize cells transfecting with a DNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Also, some proteins are recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vectors, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids.

Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol, 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): e141, both hereby incorporated by reference in their entirety.

A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5′ phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: amp^(r), cam^(r), tet^(r), blasticidin^(r), neo^(r), hyg^(r), abx^(r), neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP-glucose:galactose-1-phosphate uridyltransferase I (galT), feedback-insensitive a subunit of anthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR), benzyladenine-N-3-glucuronide, E. coli threonine deaminase, glutamate 1-semialdehyde aminotransferase (GSA-AT), D-amino acid oxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSB 1), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonia-lyase (dsdA).

In certain embodiments, sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. For example, the polypeptides GGGGGG (SEQ ID NO: 24) and GGGGT (SEQ ID NO: 25) have a sequence identity of 4 out of 5 or 80%. For example, the polypeptides GGGPPP (SEQ ID NO: 26) and GGGAPPP (SEQ ID NO: 27) have a sequence identity of 6 out of 7 or 85%. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity. Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic—F Y W; hydrophobic—A V I L; Charged positive: R K H; Charged negative—D E; Polar—S T N Q.

“Specifically binds” refers to the ability of a specific binding agent (such as an VLR or fragment thereof) of the present disclosure to recognize and bind mature, full-length or partial-length target polypeptide (herein tumor associated antigen), or an ortholog thereof, such that its affinity (as determined by, e.g., Affinity ELISA or assays as described herein) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity of the same for a random polypeptide of similar overall hydrophobicity.

A “label” refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a “label receptor” refers to incorporation of a heterologous polypeptide in the receptor. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

Variable Lymphocyte Receptor

The jawless vertebrates, lamprey and hagfish, have an adaptive immune system composed of clonally diverse lymphocytes that express variable lymphocyte receptors (VLRs). The germ-line VLRB gene is incomplete, consisting of invariant 5′ and 3′ constant regions separated by a non-coding intervening sequence. The 5′ constant region encodes a signal peptide and part of the N-terminal LRR (LRR-NT). The 3′ constant region encodes for a portion of the LRR-CT and an invariant stalk region. The incomplete germ-line gene is flanked by hundreds of partial LRR gene segments. In developing lymphocytes, the flanking LRR gene segments are randomly and sequentially copied into the incomplete VLRB gene. As each LRR gene segment is copied into the locus, it replaces a portion of the intervening sequence. The assembly mechanism continues until all of the intervening sequence is replaced with LRR modules and a functional VLRB is expressed. VLRB gene assembly occurs on only one allele, such that each lymphocyte expresses one VLRB gene.

The mature VLRB gene encodes for a crescent-shaped protein, with amino acid sequence diversity concentrated on the concave surface. The concave surface is composed of parallel β-strands and a C-terminal variable loop. Each LRR subunit contributes one β-strand, and each β-strand has five variable amino acid positions. VLRB antibodies also have variable numbers of LRR subunits. The smallest VLRB antibodies have 4 LRR subunits and the largest have 11 LRR subunits. Each LRR subunit increases the curvature of the concave surface and increases the concave surface area. The C-terminal LRR, LRR-CT, encodes a loop of variable length and sequence composition that projects above the concave surface. Immunization with particulate antigens, such as Bacillus anthracis exosporium or human red blood cells (RBCs), induces antigen-binding VLRB+ cells to proliferate and differentiate into plasmacytes. The plasmacytes secrete multivalent VLRB antibodies that circulate in the blood. Each secreted VLRB antibody is composed of identical VLRB polypeptide chains arranged into a pentamer or tetramer of dimers that is held together by disulfide bonds at the C-terminus of the flexible, invariant stalk region. Due to this multivalency, VLRB antibodies bind to their antigens with high avidity.

VLRB cDNAs expressed in mammalian cells lines (HEK-293T and CHO cells) are secreted into the tissue culture supernatant as disulfide-linked, multivalent antibodies, like VLRB in vivo. To isolate antigen-specific VLRB clones, a VLRB cDNA library is prepared from the lymphocytes of immunized lampreys. The VLRB cDNAs are transfected into HEK-293T, and the VLRB-containing tissue culture supernatants are screened for antigen binding.

Yu et al. report the generation of panels of monoclonal VLR antibodies from lamprey larvae immunized with human T cells and the use of a recombinant monoclonal VLR antibody for antigen purification and mass spectrometric identification. See J Immunol Methods, 2012, 386(1-2):43-9 entitled “Purification and identification of cell surface antigens using lamprey monoclonal antibodies.”

In certain embodiments, contemplated recombinant vectors comprise a nucleic acid that encodes a chimeric polypeptide comprising a targeting sequence of variable lymphocyte receptor domain or variant thereof, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain.

Variable lymphocyte receptors typically contain an N-terminal LRR sequence, a C-terminal LRR sequence, and multiple interior LRR modules of approximately 12-25 amino acids. The C-terminal LRR sequence typically contains a variable loop (highly variable insert). Within the interior LRR modules, seven amino acids typically contain one or two leucine or isoleucine, if two, separated by a single amino acid, e.g., (SEQ ID NO: 1) XXLXLXX, that are typically located on the concave surface, wherein X may be any amino acid and L may be leucine or isoleucine. In some instants, one L (leucine or isoleucine) may be substituted with any amino acid.

In certain embodiments, the variable lymphocyte receptor domain contains a polypeptide sequence of less than 250 amino acids and 4 or 5 or more segments having the sequence XXLXLXX (SEQ ID NO: 1) wherein X may be any amino acid and L may be, individually and independently at each occurrence, leucine or isoleucine or one L (leucine or isoleucine) may be substituted with any amino acid.

In certain embodiments, the variable lymphocyte receptor has a sequence VXCXXXXLXS VPAXIPTTTXXLXXXXNXITKXXPGVFDXLXXLXXXXLXXNXLXXXPXGXFD (SEQ ID NO: 2) wherein X may be any amino acid.

In certain embodiments, any of the variable lymphocyte receptors variants disclosed herein are considered to be those that have an altered amino acid sequence, e.g., amino acid substitutions, deletions, insertions, or combinations thereof wherein the altered sequence maintains the ability to specifically bind the antigen of interest. In some embodiment, the substitutions, deletions, insertions are or are not within the 4 or 5 segments having SEQ ID NO: 1 or a segment having SEQ ID NO: 2.

In certain embodiments, the variants contain 1, 2, or 3, amino acid substitutions. In certain embodiments, the variants of SEQ ID NO: 1, 2, 4, 6, 8, or 10, contain 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiment, the substitutions are or are not within the 4 or 5 segments having SEQ ID NO: 1. In certain embodiments, the variant substitutions are conserved substitutions. In certain embodiments, the amino acids are conserved substitutions if they are among a group with similar properties according to the following amino acid groups: Aromatic—F Y W; hydrophobic—A V I L; Charged positive—R K H; Charged negative—D E; Polar—S T N Q.

In certain embodiments, the variants contain 1, 2, or 3, amino acid deletions. In certain embodiments, the variants of SEQ ID NO: 1, 2, 4, 6, 8, or 10, contain 4, 5, 6, 7, 8, 9, or 10 amino acid deletions. In certain embodiments, the deletions are terminal deletions, e.g., starting from the first N-terminal amino acid inward or the last C-terminal amino acid. In certain embodiments, the deletions are interior deletions, e.g., between the first N-terminal amino acid identified in the SEQ ID NO and the last C-terminal amino acid. In certain embodiments, the deletions are not within the 4 or 5 segments having SEQ ID NO: 1.

In certain embodiments, the variants contain 1, 2, or 3, amino acid additions. In certain embodiments, the variants of SEQ ID NO: 1, 2, 4, 6, 8, or 10, contain 4, 5, 6, 7, 8, 9, or 10 amino acid additions. In certain embodiments, the additions are terminal additions, e.g., starting from the first N-terminal amino acid outward or the last C-terminal amino acid. In certain embodiments, the additions are interior deletions, e.g., between the first N-terminal amino acid identified in the SEQ ID NO and the last C-terminal amino acid. In certain embodiments, the deletions are not within the 4 or 5 segments having SEQ ID NO: 1.

A skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques. In certain embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In certain embodiments, one can identify residues and portions of the molecules that are conserved among similar polypeptides. In certain embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues which are important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change may be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

Variants of individual variable lymphocyte receptor may be isolated from combinatorial libraries using protein display technologies, e.g., phage display, yeast surface display, bacterial display, or cell-free systems. See Finlay et al., Methods Mol. Biol., 2011, 681:87-101; Daugherty, Curr. Opin. Struct. Biol., 2007, 17:474-480; Gai & Wittrup, Curr. Opin. Struct. Biol., 2007, 17: 467-473; Zhou et al., MAbs., 2010, 2:508-518; Shen et al., Proc. Nat. Acad. Sci. USA, 2005, 102: 5969-5974. Typically, a collection of unique variants are linked through the display platform by expression from corresponding mutated nucleic acids. After exposing/mixing the variants expressed on the display platform with the target molecule, molecule bound variants are identified and/or separated for analysis. Typically, the protein sequence is determined by sequencing an associated nucleic acid in the display platform. For example, in yeast surface display, recombinant yeast cells express variant proteins wherein the yeast cell expresses the variant protein conjugated to a cell wall protein. The yeast cell can contain a plasmid DNA that encodes the variant protein which is expressed on the surface of the yeast cell and sequencing the plasmid DNA provides the protein sequence of the variant protein. See Gera et al., Protein selection using yeast surface display, Methods, 2013, 60(1):15-26. In phage display, variant proteins are typically conjugated to a bacteriophage coat protein. Cell-based systems also typically rely on the expression of variant proteins conjugated to cell surface proteins, e.g., in bacterial, yeast, and mammalian cells, and the host cell carries a plasmid vector that encodes the variant proteins. Cell-free systems have also been developed wherein the variant protein is conjugated directly to its encoding mRNA, termed ribosome display or mRNA display.

In certain embodiments, the disclosure relates to isolated polypeptides comprising SEQ ID NO: 4, 6, 8, 10, or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a polypeptide disclosed herein or chimeric protein thereof.

In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col E1 origin of replication, f1 origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

Immunotherapy and Chimeric Antigen Receptors

In order to improve the ability of immune cells to kill cancerous cells, T cells can be isolated from the blood of a patient and genetically altered to specifically target proteins expressed on the surface of cancerous cells. When put back into the patient, the cells can be more efficient binders of the cancerous cells. CD19 is a protein expressed on cancerous B cells. Brentjens et al. report that T cells altered to bind CD19 can induce remissions of cancer in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med, 2013, 5(177):177ra38.

In certain embodiments, the disclosure relates to recombinants vector comprising a nucleic acid that encodes a chimeric polypeptide comprising a targeting sequence of variable lymphocyte receptor domain or variant thereof, a transmembrane domain, a T cell costimulatory molecule domain, and a signal-transduction component of a T-cell antigen receptor domain.

In certain embodiments, the targeting sequence is a variable lymphocyte receptor domain or any variety of polypeptide sequences capable of selectively binding to a surface protein on target cells, e.g., cancer cells. Other targeting sequences may be variable binding regions of antibodies, single chain antibodies, and antibody mimetic.

In certain embodiments, the costimulatory molecule is selected from CD28 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGL DSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVM YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 11) or variants or fragment thereof such as the immunoglobulin (Ig) domain of cytotoxic T lymphocyte-associated antigen 4 ILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQV YSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVK (SEQ ID NO: 12) or variants or fragment thereof. CD28 is the receptor for CD80 (B7.1) and the disclosure contemplates CD80 as the sequence MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEEL AQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVV LKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGE ELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHF PDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 13) or variants or fragment thereof variants or a sequence with greater than 50, 60, 70, 80, 90, 95% or greater identity thereto. CD28 is also the receptor for CD86 (B7.2) and the disclosure contemplates CD86 as the sequence MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQD QENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPT GMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTI EYDGIIVIQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPP PDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIH IPERSDEAQRVFKSSKTSSCDKSDTCFP (SEQ ID NO: 14) or variants or fragment thereof variants or a sequence with greater than 50, 60, 70, 80, 90, 95% or greater identity thereto.

In certain embodiments, the disclosure contemplates using a co-stimulating molecule that is a 20 to 100 or 50 to 150 amino acid fragment of SEQ ID NO: 11-14 or variants thereof or those with 80, 90, 95% or greater identity thereto.

In certain embodiments, the signal-transduction component of the T-cell antigen receptor is a peptide with an immunoreceptor tyrosine-based activation motif with the consensus sequence YXXLXXXXXXXXYXXL (SEQ ID NO: 15) wherein X is any amino acid L is leucine or isoleucine, wherein SEQ ID NO: 15 optionally has one or two X amino acid deletions within the middle segment XXXXXXXX (SEQ ID NO: 16). The immunoreceptor tyrosine-based activation motif (underlined) is in the partial CD3-zeta sequence

(SEQ ID NO: 17) AQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPA YQQGQNQL YNELNLGRREEYDVL DKRRGRDPEMGGKPQRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R

or fragments or variants, e.g. having 1, 2, or 3 amino acid deletion, addition, or substitution variants, or a sequence with greater than 50, 60, 70, 80, 90, 95% or greater identity thereto.

In certain embodiments, the signal-transduction component of the T-cell antigen receptor is a peptide with a immunoreceptor tyrosine-based activation motif (underlined) with the sequence of immunoglobulin epsilon receptor subunit gamma precursor

(SEQ ID NO: 18) IPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVR KAAITSYEKSDGV YTGLSTRNQETYETL KHEKPPQ

fragments or variants thereof variants or a sequence with greater than 50, 60, 70, 80, 90, 95% or greater identity thereto.

In certain embodiments, the disclosure relates to recombinant vectors comprising a nucleic acid that encodes a chimeric polypeptide as provided herein further comprising an interleukin sequence such as a human IL-2 signal sequence (amino acids 1-60 of IL-2) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 19), fragments, variants or a sequence with greater than 50, 60, 70, 80, 90, 95% or greater identity thereto.

In certain embodiments, the disclosure relates to recombinant vectors comprising a nucleic acid that encodes a chimeric polypeptide as provided herein further comprising a human CD8 sequence MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQ PRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSAL SNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 20) or variant or fragment with a sequence with 50, 60, 70, 80, 90, 95% or greater identity thereto.

In certain embodiments, the disclosure relates to recombinant vectors comprising a nucleic acid that encodes a chimeric polypeptide as provided herein further comprising a human CD137 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR TCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAP AREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCEL (SEQ ID NO: 21) or variant or fragment with a sequence with 50, 60, 70, 80, 90, 95% or greater identity thereto

Immunotherapy

In certain embodiments, the disclosure relates to methods of treating cancer comprising isolating T cells, gamma delta T cells, or NK cells and transferring a recombinant vector of disclosed herein into the cells under conditions such that the recombinant vector expresses in the isolated cells a chimeric polypeptide comprising a targeting domain, e.g., variable lymphocyte receptor domain or variant, a transmembrane molecule domain, a T cell co-stimulatory molecule domain, and a signal-transduction component of the T-cell antigen receptor domain providing modified T cells, gamma delta T cells, or NK cells; and implanting the modified T cells, gamma delta T cells, or NK cells into a subject in need thereof.

In certain embodiments, the disclosure relates to recombinants vector comprising a nucleic acid that encodes a chimeric polypeptide comprising a targeting sequence, a transmembrane domain, a T cell co-stimulatory molecule domain, a signal-transduction component of a T-cell antigen receptor domain, and an enzyme that confers resistance to cellular damage in the presence of a chemotherapy agent.

In certain embodiments, the recombinant vector encodes an enzyme that confers resistance to cellular damage in the presence of a chemotherapy agent, and an effective amount of the chemotherapy agent is administered to the subject before, during, or after implanting the cells into the a subject.

Immunocompetent cells exhibit cytotoxicity toward cancer cells and tumorigenic animal models have shown that these cells can infiltrate tumors resulting in tumor regression. Tumor infiltration by immunocompetent cells indicates favorable prognosis in various cancers, such as melanoma, colon, ovarian cancer, basal cell carcinoma, and lung cancer.

Chemotherapy regimens frequently lead to non-specific cellular toxicity to adoptively transferred immunocompetent cells and to hematopoietic stem cells. One strategy to combat drug-induced toxicity is to genetically engineer immune cells to make them drug resistant, e.g., using either drug resistant bone marrow or immunocompetent cells with intrinsic cytotoxic capabilities. Several genes, such as methylguanine methyltransferase (MGMT), dihydrofolate reductase (DHFR), cytidine deaminase (CD), and multidrug resistant protein (MDR-1) can confer drug resistance to anti-cancer immune cells. Recombinant retroviral vectors, such as lentiviral vectors, are efficient gene transfer systems for the ex vivo modification of cells, e.g., hematopoietic cells. Retroviral gene transfer results in integration of the transferred nucleic acid sequence into the genome of the target cell.

In certain embodiments, the disclosure contemplates recombinant vectors encoding P140KMGMT polypeptide MDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAA DAVEVPAPAAVLGGPEPLMQCTAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVL WKLLKVVKFGEVISYQQLAALAGNPKAARAVGGAMRGNPVKILIPCHRVVCSSGAVG NYSGGLAVKEWLLAHEGHRLGKPGLGGSSGLAGAWLKGAGATSGSPPAGRN (SEQ ID NO: 22) and/or L22YDHFR polypeptide VGSLNCIVAVSQNMGIGKNGDYPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKTWF SIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGS SVYKEAMNHPGHLKLFVTREVIQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEKND (SEQ ID NO: 23) or a sequence with greater than 80, 90 or 95% identity thereto.

Zielske et al. report lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors confers significant resistance to BG/BCNU and allows selection in vitro. Mol Ther. 2002, 5(4):381-7. Sawai et al. report protection and in vivo selection of hematopoietic stem cells using temozolomide, 06-benzylguanine, and an alkyltransferase-expressing retroviral vector. Mol Ther. 2001, 3(1):78-87. Maier et al. report F2A sequence linking MGMT(P140K) and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of hematopoietic stem cells. Cancer Gene Ther. 2012, 19(11):802-10.

In certain embodiments the immunocompetent cells, e.g., isolated T cells, gamma delta T cells, or NK cells are isolated from the subject to receive the implanted modified cells.

In certain embodiments, the cancer is selected from neuroblastoma, glioblastoma, glioma, breast cancer, prostate cancer, colon cancer, lung cancer, skin cancer, renal cancer, ovarian cancer, pancreatic cancer, stomach cancer, leukemia, lymphoma, or melanoma.

In certain embodiments, the targeting sequence specifically binds to a tumor associated antigen such as CD5, CD19, CD20, CD30, CD33, CD47, CD52, CD152(CTLA-4), CD274(PD-L1), CD340(ErbB-2), GD2, TPBG, CA-125, CEA, MAGEA1, MAGEA3, MART1, GP100, MUC1, WT1, TAG-72, HPVE6, HPVE7, BING-4, SAP-1, immature laminin receptor, vascular endothelial growth factor (VEGF-A) or epidermal growth factor receptor (ErbB-1).

In certain embodiments, the disclosure relates to an isolated cell comprising the recombinant vectors disclosed herein. In certain embodiments, the isolated cells are selected from T helper cells, cytotoxic T cells, natural killer T cells, or gamma delta T cells. These cells may be obtained by isolation from peripheral blood and optionally purified by fluorescent activated cells sorting e.g., mixing cells with fluorescent antibodies or other fluorescent agents and separating the cells by flow cytometry based fluorescent sorting. Another option for cells sorting is to provide magnetic particles that are conjugated to antibodies against a particular antigen on the cell surface.

After mixing with a sample, the antibody bound cells are put through a purification column containing a matrix composed of ferromagnetic spheres. When placed on a magnetic separator, the spheres amplify the magnetic field. The unlabeled cells pass through while the magnetically labeled cells are retained within the column. The flow-through can be collected as the unlabeled cell fraction. After a short washing step, the column is removed from the separator, and the magnetically labeled cells are eluted from the column.

CD3 is expressed on all T cells as it is associated with the T cell receptor (TCR). The majority of TCR are made up of alpha beta chains (alpha beta T-cells). Alpha beta T-cells and gamma delta T cells are believed to be derived from a common CD4⁻CD8⁻ double-negative precursor thymocytes. Mature gamma delta T cells are CD4⁻CD8⁻ double-negative. In contrast, alpha beta T-cells typically become double-positive intermediates (CD4⁺CD8⁺) which mature into single-positive (CD4⁺CD8⁻) T helper cells or (CD4⁻CD8⁺) cytotoxic T cells. Memory cells may be either CD4⁺ or CD8⁺. Memory T cells typically express the cell surface protein CD45RO. T cells may be isolated and separated from a human sample (blood or PBMCs) based on the expression of alpha beta T cell receptor (TCR), gamma delta T cell receptor, CD2, CD3, CD4, CD8, CD4 and CD8, NK1.1, CD4 and CD25 and other combinations based on positive or negative selection. TCRγ/δ⁺ T cells are TCRα/β−, CD2⁺, CD3⁺, and CD5⁺ See also Salot et al., Large scale expansion of Vgamma9Vdelta2 T lymphocytes from human peripheral blood mononuclear cells after a positive selection using MACS “TCR gamma/delta⁺ T cell isolation kit,” J Immunol Methods, 2009, 347(1-2):12-8.

In certain embodiments, the disclosure contemplates methods of engineered immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes comparing a VLR with a targeting tumor associated antigen/molecule.

In certain embodiments, the molecule is a tumor associates molecule selected from CD20, CD20, CD30, CD33, CD52, EpCAM, epithelial cell adhesion molecule, gpA33, glycoprotein A33, Mucins, TAG-72, tumor-associated glycoprotein 72, Folate-binding protein, VEGF, vascular endothelial growth factor, integrin αVβ3, integrin α5β1, FAP, fibroblast activation protein, CEA, carcinoembryonic antigen, tenascin, Le, Lewis Y antigen, CAIX, carbonic anhydrase IX, epidermal growth factor receptor (EGFR; also known as ERBB1), ERBB2 (also known as HER2), ERBB3, MET (also known as HGFR), insulin-like growth factor 1 receptor (IGF1R), ephrin receptor A3 (EPHA3), tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAILR1; also known as TNFRSF10A), TRAILR2 (also known as TNFRSF10B) and receptor activator of nuclear factor-KB ligand (RANKL; also known as TNFSF11) and fragments thereof.

In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with breast cancer, prostate cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, bladder cancer, brain cancer, kidney cancer, endometrial cancer, pancreatic cancer, and thyroid cancer.

In certain embodiments, contemplated methods include further administering a second anti-cancer agent such as bevacizumab, gefitinib, erlotinib, temozolomide, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, idoxifene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.

In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of one more other anti-cancer agents. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of trastuzumab and/or lapatinib. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of docetaxel and cyclophosphamide. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of docetaxel, carboplatin, and trastuzumab. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of cyclophosphamide, doxorubicin, and 5-fluorouracil (5-FU). In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of docetaxel, doxorubicin, and cyclophosphamide. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of doxorubicin and cyclophosphamide followed by paclitaxel or docetaxel. In certain embodiments, the disclosure contemplates treating or preventing breast cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of 5-FU, epirubicin, and cyclophosphamide followed by docetaxel or paclitaxel.

In certain embodiments, the disclosure contemplates treating or preventing prostate cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of one more other anti-cancer agents. In certain embodiments, the disclosure contemplates treating or preventing prostate cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of leuprolide, goserelin, or buserelin. In certain embodiments, the disclosure contemplates treating or preventing prostate cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of flutamide, bicalutamide, enzalutamide, or nilutamide. In certain embodiments, the disclosure contemplates treating or preventing prostate cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of ketoconazole or aminoglutethimide. In certain embodiments, the disclosure contemplates treating or preventing prostate cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of abiraterone, bicalutamide, cabazitaxel, bicalutamide, degarelix, denosumab, docetaxel, enzalutamide, cabazitaxel, leuprolide, prednisone, denosumab, sipuleucel-T, or radium 223 dichloride and combinations thereof.

In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of one more other anti-cancer agents. In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of 5-FU, leucovorin, or capecitabine or combinations thereof. In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of capecitabine and oxaliplatin. In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of 5-FU, leucovorin, and oxaliplatin. In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of leucovorin, 5-FU, and irinotecan. In certain embodiments, the disclosure contemplates treating or preventing colon cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of leucovorin, 5-FU, oxaliplatin, and irinotecan.

In certain embodiments, the disclosure contemplates treating or preventing lung cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of vinorelbine, etoposide, mitomycin C, gemcitabine, irinotecan, pemetrexed, gefitinib, erlotinib, lapatinib, crizotinib, and a vinca alkaloid or combinations thereof. In certain embodiments, the vinca alkaloid is vinblastine, vincristine, vindesine, or vinorelbine. In certain embodiments, the disclosure contemplates treating or preventing lung cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of bevacizumab panitumumab, zalutumumab, nimotuzumab, matuzumab, or cetuximab. In certain embodiments, the disclosure contemplates treating or preventing lung cancer using methods of modified immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with the administration of a platinum-based agent and/or a taxane e.g., paclitaxel and docetaxel or combinations thereof.

In certain embodiments, the disclosure contemplates treating or preventing brain cancer, glioblastoma multiforme, oligodendroglioma, primitive neuroectodermal tumors, ependymomas, or glioma. In certain embodiments, the chimeric protein is optionally administered in combination with temozolomide, procarbazine, carmustine (BCNU), lomustine (CCNU), vincristine, and combinations thereof. In certain embodiments, procarbazine, lomustine (CCNU) and vincristine are combined. In certain embodiments, the chimeric protein is optionally administered in combination with irinotecan, cis-platin, carboplatin, methotrexate, etoposide, bleomycin, vinblastine, actinomycin (Dactinomycin), cyclophosphamide, or ifosfamide.

In certain embodiments, the disclosure contemplates methods combining engineered immune cells treatments disclosed herein, such as those wherein T cells are engineered to express drug resistant enzymes, in combination with temozolomide treatments. Treatment of glioblastoma includes chemotherapy during and after radiotherapy. On average, chemotherapy after surgery and radiotherapy can initially reduce the tumor size.

EXPERIMENTAL

Creation of VLR-CAR Sequences that Bind to Tumor Cells/Neuroblastoma

Naïve lampreys were serially immunized with intact cells (for example, human T cells, murine B cell leukemia, human neuroblastoma cell line) over 6 weeks. Subsequently, their lymphocytes were harvested, and RNA was obtained and converted to cDNA. PCR amplification of the VLR sequences from the cDNA library facilitated cloning of the VLRs into a yeast surface expression library which was used to screen for VLRs that bind to antigens on the target cells by flow cytometry (See FIGS. 3 and 4 illustrate the process used to generate VLR sequences that can be used to target tumor cells.) A similar high throughout process incorporating normal cells or tissue can be used to negatively select VLRs that are likely to generate off-target CAR effects. VLRs meeting the set criteria then are sequenced and cloned into a CAR transgene cassette. The VLR-CAR was subsequently cloned into a lentiviral vector, which was used to produce high titer recombinant lentiviurs. The recombinant virus was used to transduce T cells (Jurkat cells). As shown in FIG. 4, anti-neuroblastoma VLR-CAR (generated against the neuroblastoma tumor cell line, SK—N—Be(2)) effectively activated T cells in the presence of stimulatory cells.

VLR Sequences that Bind to B-Cell Leukemia

A CAR was developed that contains a VLR specific for the B-cell receptor of a murine B cell leukemia (BCL) cell line. The CAR design incorporates the anti-BCL-VLR, a Myc tag, CD28 transmembrane domain, and the intracellular CD3ζ signaling domain (FIG. 5). SIN VLR-CAR lentiviral vector was produced at high titer (˜1×10⁸) and used to transduce HEK 293T and Jurkat cells. Transduced Jurkat cells demonstrated persistent surface CAR (FIGS. 6A-C) without overt toxicity. To determine whether the VLR was capable of signaling through the CAR, the transduced Jurkat cells were incubated with the BCL cell line expressing the target B-cell receptor. Using this assay, potent T cell activation via the VLR-CAR was demonstrated (FIGS. 7A-C).

VLR Sequences that Bind to T-Cell Leukemia

Yu, C., et al., 2012 identified a VLR sequence that recognizes CD5, which is present, for example, on T cell leukemias. A codon optimized cDNA was synthesized that encodes the VLR and was cloned into the CAR sequence shown in FIG. 5 in place of the BCL-VLR. High titer virus was generated and used to transduce T cells, similar to the studies described above. Cells transduced with a GFP expressing cassette were not activated in the presence of CD5 expressing cells, but cells transduced with the CD5 VLR-CAR showed increased CD69 expression (FIG. 8A), indicating T cell activation. 

The invention claimed is:
 1. A method of treating neuroblastoma comprising isolating T cells, gamma delta T cells, or NK cells and transferring a recombinant vector into the cells under conditions such that the recombinant vector expresses in the isolated cells a chimeric polypeptide comprising a variable lymphocyte receptor domain, wherein the variable lymphocyte receptor has SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, a transmembrane molecule domain, a T cell costimulatory molecule domain, and a signal-transduction component of the T-cell antigen receptor domain providing modified T cells, gamma delta T cells, or NK cells; and implanting an effective amount of the modified T cells, gamma delta T cells, or NK cells into a subject in need thereof.
 2. The method of claim 1, wherein the costimulatory molecule is selected from CD28, CD80, CD86 or variant or fragment thereto.
 3. The method of claim 1, wherein the signal-transduction component of the T-cell antigen comprises sequence YXXLXXXXXXXXYXXL (SEQ ID NO: 15) wherein X is any amino acid, L is leucine or isoleucine, and one or two X are optionally deleted.
 4. The method of claim 1, wherein the chimeric polypeptide further comprises an interleukin signal sequence.
 5. The method of claim 4, wherein the interleukin signal sequence is IL-2.
 6. The method of claim 1, wherein the chimeric polypeptide further comprises CD8.
 7. The method of claim 1, wherein the isolated T cells, gamma delta T cells, or NK cells are isolated from the subject to receive the implanted modified cells.
 8. A method of treating a CD5 positive cancer comprising isolating T cells, gamma delta T cells, or NK cells and transferring a recombinant vector into the cells under conditions such that the recombinant vector expresses in the isolated cells a chimeric polypeptide comprising a variable lymphocyte receptor domain, wherein the variable lymphocyte receptor has SEQ ID NO: 10 encoded by SEQ ID NO: 9, a transmembrane molecule domain, a T cell costimulatory molecule domain, and a signal-transduction component of the T-cell antigen receptor domain providing modified T cells, gamma delta T cells, or NK cells; and implanting an effective amount of the modified T cells, gamma delta T cells, or NK cells into a subject in need thereof.
 9. The method of claim 8, wherein the costimulatory molecule is selected from CD28, CD80, CD86 or variant or fragment thereto.
 10. The method of claim 8, wherein the signal-transduction component of the T-cell antigen comprises sequence YXXLXXXXXXXXYXXL (SEQ ID NO: 15) wherein X is any amino acid, L is leucine or isoleucine, and one or two X are optionally deleted.
 11. The method of claim 8, wherein the chimeric polypeptide further comprises an interleukin signal sequence.
 12. The method of claim 11, wherein the interleukin signal sequence is IL-2.
 13. The method of claim 8, wherein the chimeric polypeptide further comprises CD8.
 14. The method of claim 8, wherein the isolated T cells, gamma delta T cells, or NK cells are isolated from the subject to receive the implanted modified cells.
 15. The method of claim 8, wherein the CD5 positive cancer is selected from neuroblastoma, glioblastoma, glioma, breast cancer, prostate cancer, colon cancer, lung cancer, skin cancer, renal cancer, ovarian cancer, pancreatic cancer, stomach cancer, leukemia, lymphoma, or melanoma. 